Hepatitis E virus (HEV) causes endemic and large epidemics of acute human hepatitis and sporadic cases by a fecal-oral transmission in many parts of the world. HEV generally infects young adults and the disease has a high mortality rate, up to 20%, in infected pregnant women. Hepatitis E may be a zoonosis because HEV infection has been found in rodents and domestic animals and direct evidence of transmission from animal to human was reported. The waterborne and zoonosis features of hepatitis E pose a public health concern. Development of specific anti-HEV drugs is urgently needed to provide treatment strategy for those who are ill with hepatitis E. The long term goal of our research is to develop specific molecular anti-HEV drugs. The objective of this project is to explore antisense phosphorodiamidate morpholino antisense oligomers (PMO) drugs to block HEV replication. The specific aims proposed are to assess antisense PMO oligomers targeting HEV with a reporter assay, determine the efficacy of the PMO on HEV replication in cells. PMO are analogs of short DNA oligomers with modified sugar and phosphate moieties in the nucleotides, resulting in high target- binding specificity and complete resistance to nuclease in the host. Their safety, efficacy and bioavailability suggest promising applications in the clinical arena. An obvious advantage of the nucleic acid-based approach for anti-HEV drugs is that target regions can be selected from HEV genomic sequences. Propagation of HEV and studies on HEV replication has relied on non-human primate model due to shortage of efficient supportive cell culture system. The recent establishment of in vitro replication of HEV genomes and HEV replicon in human hepatoma cells transfected with transcripts of infectious cDNA clone provides an additional approach to study HEV biology. The in vitro system for replication of HEV will be used to evaluate the PMO effects on HEV replication in this project. It is expected that completion of these studies will provide us with effective PMO that can inhibit HEV replication and that can be useful reagent to study HEV biology. Project narrative: This project is proposed to develop specific molecular antivirals against hepatitis E virus, the causative agent of endemic and epidemic acute human hepatitis. The antiviral compounds are expected to bind to the virus genomic sequence and suppress the virus replication.